Neuronal and retinal tissue are high in phospholipids containing either one or two polyunsaturated acyl chains. In order to better characterize the acyl chain packing properties of polyunsaturated phospholipids, we have developed a refined analysis of the time resolved anisotropy decay of free tumbling membrane probes. In these studies we have used the hydrophobic fluorescence probe diphenylhexatriene (DPH), one of the most commonly used probes. This analysis allows a measure of the acyl chain packing order in the membrane interior vs the portion of the chain closer to the interfacial region and provides complementary information to that obtained from intramolecular order parameters derived from NMR. In contrast to NMR data, which reflects the properties of individual molecules, the fluorescence data is representative of the time averaged properties of the ensemble of lipid molecules composing the bilayer. These studies demonstrate that the presence of polyunsaturated acyl chains results in the highest degree of acyl chain disorder and produce the highest values of acyl chain packing free volume. This latter parameter has been shown to promote the greatest degree of formation of the activate conformation of the G protein-coupled receptor, rhodopsin. Cholesterol has been shown to decrease both acyl chain packing free volume and rhodopsin activation. Our studies show that polyunsaturated phospholipids are able to resist the ordering effects of cholesterol and maintain their unique acyl chain packing properties. These findings have important implications for integral membrane protein function and suggest that highly unsaturated phospholipids found in retina and neuronal tissue can optimize receptor function and are able to resist the effects of certain compositional variation, such as increases in cholesterol content.